Dipeptidylaminopeptidase I (DAP 1) (EC 3.4.14.1), or cathepsin C, has long been investigated in term of its purification, kinetic mechanism, and physiological roles. It is a member of a group of lysosomal cysteine proteases involved in protein degradation. It is a dipeptidylaminopeptidase which removes N-terminal dipeptides sequentially from an unsubstituted N-terminal peptide or protein with broad substrate specificity. It has been postulated to function in protein turnover. DAP 1 absence/overproduction has been proposed to be involved in Duchenne muscular dystrophy. C. N. Pato, et. al., Proceedings of the National Academy of Sciences, U.S.A., 80:4732-4736 (1983). DAP 1 has been demonstrated to be present in elevated levels in cytotoxic lymphocytes. D. L. Thiele and P. E. Lipsky, Proceedings of the National Academy of Sciences, U.S.A., 87:83-87 (1990).
DAP 1 has been isolated from a variety of sources including bovine spleen [R. M. Metrione, et. al., Biochemistry, vol. 5 (1990)], bovine pituitary [J. K. McDonald, et. al., Journal of Biological Chemistry, 241:1494-1501 (1966)], rat liver [F. L. Huang and A. L. Tappel, Biochimica et Biophysica Acta, 268:527-538 (1972)] and human spleen [M. McGuire, et al., Archives of Biochemica et Biophysica, 295:280-288 (1992)]. It consists of several polypeptides in a complexed oligomeric structure. R. M. Metrione, et al., Biochemistry, vol. 9 no. 12.
In the case of bovine spleen DAP 1, the purified enzyme has three distinctive subunits: 23 kD (a chain), 21 kD (b chain), 5.6 kD (g chain), forming an oligomeric structure of a4b4g4. It has been reported that the subunits of rat DAPI may be derived from a single precursor as evidence in rat cell lines (F. Mainferme, et al., European Journal of Biochemistry, 153: 211-216 (1985); and V. Burge, et al., Biochemistry Journal, 275:707-800 (1991). Recent cloning experiments have confirmed that the oligomeric protein is actually a proteolytic product of a single polypeptide. K. Ishidoh, et al., Journal of Biological Chemistry, 266:16312-16317 (1991).
In addition to bovine dipeptidylaminopeptidase 1 described in the publications above, another dipeptidylaminopeptidase frequently employed in the processing of recombinant proteins is that derived from the slime mold Dictyostelium discoidium. The synthesis, purification and use of this protease, often abbreviated as dDAP, are described in European Patent Publication 595,476, published May 4, 1994, and U.S. patent application Nos. 08/301,519, filed Sep. 7, 1994, and 08/445,308, filed May 19, 1995, all of which are herein incorporated by reference.
Bovine DAP 1 has several unique properties. First, it is a thiolproteinase instead of being a serine proteinase as is a class of dipeptidylaminopeptidases, such as DAPII, DAPIII, and DAPIV. Second, it functions as a dipeptidylaminopeptidase in a thiolproteinase family which are endopeptidases. Third, it strongly requires Cl.sup.- for maximal activity and is mildly heat resistant. J. Gorter and M. Gruber, Biochimica et Biophysica Acta, 198:546-555 (1970). The enzyme is beginning to become one of the major bioprocessing enzymes used in industry.
Until the present invention, it was not known whether the cDNA sequence encoding bovine DAP 1 consisted of three independent cDNAs or was encoded in by a single cDNA. The present invention demonstrates that bovine DAP 1 is encoded by a single cDNA. Due to the increasing importance of avoiding infectious agents, be they viruses or prions, arising from animal sourced material, the pharmaceutical industry has favored the use of non-animal sourced enzymes in the biosynthetic processing of pharmaceutical proteins. Recombinant expression of such enzymes has thus been favored. It is, therefore, advantageous to have a cloned source for the enzyme.